Monoclonal antibody hybridoma immunoassay method and diagnosis kit

ABSTRACT

The object of the present invention is to provide a diagnostic method by which  Helicobacter pylori  infection can be diagnosed at low cost without causing pain on subjects and without requiring particular equipment and which is free of cross reactivity and excellent in specificity without variation among lots as a result of the use of a single antibody facilitating the quality control and which shows good sensitivity even when a single monoclonal antibody is used. 
     The present invention provides a monoclonal antibody which recognizes  Helicobacter pylori  catalase as an antigen.

This is a National Stage Entry under 35 U.S.C. § 371 of PCT ApplicationNo. PCT/JP00/07554, filed Oct. 27, 2000.

TECHNICAL FIELD

The present invention relates to a monoclonal antibody recognizingHelicobacter pylori catalase as an antigen, a hybridoma producing saidmonoclonal antibody, an immunological assay method and a diagnosis kit.

BACKGROUND ART

Helicobacter pylori is a bacterial species found in the human gastricmucosa. The rate of Helicobacter pylori infection is closely related tosocial and economic factors, tending to be high in developing countriesand low in advanced countries. However, the rate of infection among theJapanese is remarkably high among the advanced countries and it is evenreported that 80% of people in and after the fourth decade of life havebeen infected. In recent years, it has been revealed that Helicobacterpylori may cause various gastric and duodenal diseases such as gastriculcer, duodenal ulcer, chronic gastritis and, further, gastric cancer.

Since it was proven that the possibility of being afflicted by thesediseases can be reduced by eradicating Helicobacter pylori,international discussions have been made about the diseases at whichHelicobacter pylori eradication is to target. Thus, the diseasescurrently considered to be targets of such eradication include gastriculcer, duodenal ulcer, malignant gastric lymphoma, residual stomachafter resection due to early gastric cancer.

With the recognition of Helicobacter pylori eradication therapy as anovel method for the treatment of gastric and duodenal diseases,guidelines for therapeutic trials have been laid down and methods fordiagnosis about occurrence of Helicobacter pylori infection and judgmentabout bacterial eradication have been proposed by the Japanese Societyof Gastroenterology (The Japanese Journal of Gastroenterology, vol. 96,199-207, 1999). According to the above guidelines for therapeutictrials, it is indicated that the diagnosis about an occurrence should becarried out based on an invasive test method, namely culture of a biopsyspecimen from a gastric site, microscopy and a urease test, and that thejudgment about bacterial eradication essentially requires culture of abiopsy specimen from a gastric site and microscopy and a urea breathtest, which is a noninvasive test method. In special cases, for examplein the case of a pediatric subject, it is indicated that the judgmentshould be carried out based on the combination of a test foranti-Helicobacter pylori antibodies in blood and the diagnosis about anoccurrence.

However, these test methods for Helicobacter pylori infection have thefollowing problems. The invasive test method inflicts a great deal ofpain on subjects on the occasions of gastroscope insertion andbiopsying. As for the noninvasive test methods, the pain sensation insubjects is markedly reduced but the urea breath test requires fastingprior to the test. Further, in carrying out the urea breath test, suchapparatus as a mass spectrometer, an infrared spectrophotometer, etc.are required, hence the test can be carried out only in specialinstitutions, and the resulting high cost is also a drawback. Theantibody test is not suited for the judgment about bacterial eradicationsince the antibody titer in blood remains at high levels for a long termafter eradication of the bacteria. Therefore, the advent of anoninvasive test method which may replace the above test methods and bywhich Helicobacter pylori infection can be detected directly andspecifically with high accuracy has been awaited.

In the art, isolation culture of infective bacteria from digestive tractexcreta, particularly feces, using a selection medium, has been made asa direct test method for infective bacteria in the digestive tract. Asfar as Helicobacter pylori is concerned, however, in spite of a largenumber of trials, there are few reports about the isolation, by culture,from feces. The reason may be considered that this microorganism hasalready undergone a transformation to the coccoid form, which cannot beculturally isolated, in the lower digestive tract since Helicobacterpylori undergoes, in vitro, a morphological change from the ordinaryhelical form to the coccoid form, which cannot be cultured, underunfavorable environmental conditions such as low temperature,nutritional deficiency and oxygen deficiency.

On the other hand, as regards direct detection of Helicobacter pylorifrom feces by an immunological method based on the antigen-antibodyreaction, there is a report about the detection of Helicobacter pyloriin excreta specimens, such as feces, by an immunoassay using polyclonalantibodies against Helicobacter pylori (J. Clin. Microbiol., vol. 33,2162-2165, 1995; Japanese Kokai Publication Hei-10-10128 (JP 3043999)).

However, polyclonal antibodies generally have cross reactivity and areinferior in specificity and, in addition, are disadvantageous in thatthe antibody titer and specificity may vary depending on the lot ofantiserum. Therefore, there is a problem that the production ofdiagnostic tests in which polyclonal antibodies are used is essentiallydifficult in terms of the quality control. In actuality, as regards thekit “HpSA” for detecting the antigen of Helicobacter pylori in feces,which is a product of Meridian, the patentee of JP No. 3043999, and inwhich a polyclonal antibody is used, questions about encountered falsepositive cases and low specificity have been raised (Medical Tribune,4-5, Jun. 3, 1999 issue; Am. J. Gastroenterol., vol. 94, 1830-1833,1999).

On the contrary, in Japanese Kokai Publication Hei-10-10128, it isdescribed to the effect that since strains of Helicobacter pylori aresusceptible to mutation, monoclonal antibodies capable of reacting onlywith respective individual antigens are not suited for use in detectingHelicobacter pylori but polyclonal antibodies, which can react withvarious antigens or epitopes, are rather suited for the detection ofHelicobacter pylori.

SUMMARY OF THE INVENTION

In view of the above state of the art, it is an object of the presentinvention to provide a diagnostic method by which Helicobacter pyloriinfection can be diagnosed at low cost without causing pain on subjectsand without requiring particular equipment and which is free of crossreactivity and excellent in specificity without variation among lots asa result of the use of a single antibody facilitating the qualitycontrol and which shows good sensitivity even when a single monoclonalantibody is used.

The present invention provides a monoclonal antibody which recognizesHelicobacter pylori catalase as an antigen.

The invention provides a hybridoma

which produces a monoclonal antibody recognizing Helicobacter pyloricatalase as an antigen.

The hybridoma of the invention is preferably 21G2 (FERM BP-7336), 41A5(FERM BP-7337) or 82B9 (FERN BP-1338).

A monoclonal antibody which is produced by the hybridoma of theinvention constitutes an aspect of the present invention.

An immunological assay method which is carried out using at least onemonoclonal antibody species of the present invention also constitutes anaspect of the invention.

The immunological assay method of the invention is preferably carriedout using any one of the monoclonal antibody species mentioned above.

The immunological assay method of the invention is preferably used formaking judgment about Helicobacter pylori infection.

A specimen for the immunological assay method of the invention ispreferably digestive tract excreta.

The immunological assay method of the invention is preferably carriedout by an ELISA or immunochromatography technique.

A diagnosis kit which comprises at least one monoclonal antibody speciesof the invention also constitutes an aspect of the present invention.

The diagnosis kit of the invention preferably comprises any one of theabove monoclonal antibody species.

The diagnosis kit of the invention is preferably used for makingjudgment about Helicobacter pylori infection.

A specimen for the diagnosis kit of the invention is preferablydigestive tract excreta.

The diagnosis kit of the invention is preferably carried out by an ELISAor immunochromatography technique.

Catalase so referred to herein does not contain those proteins whichcorrespond to subunits resulting from denaturation, dissociation orsteric structure disentanglement by means of a denaturing agent such asSDS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the elution pattern in affinity chromatography asobserved in Example 9.

FIG. 2 illustrates the elution pattern in affinity chromatography asobserved in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the present invention is described in detail.

The monoclonal antibody of the present invention recognizes Helicobacterpylori catalase as an antigen.

The monoclonal antibody of the invention can be produced by thehybridoma according to the invention and thus can be obtainable, forexample, by the culture fluid resulting from cultivating the hybridomaof the invention. However, the method of producing the monoclonalantibodies of the invention is not particularly restricted but agenetically engineered one, for instance, falls within the scope of thepresent invention if it can specifically bind to Helicobacter pyloricatalase.

The hybridoma of the invention produces a monoclonal antibodyrecognizing Helicobacter pylori catalase as an antigen and can beobtained by subjecting spleen cells or lymph node cells of an animalimmunized with Helicobacter pylori to cell fusion with myeloma cells.

The hybridoma of the present invention can be produced by a cell fusiontechnique known in the art. Thus, an animal other than human isimmunized with Helicobacter pylori as an immunogen, hybridomas areproduced by causing spleen cells or lymph node cells of that animal tofuse with myeloma cells, and a hybridoma producing a monoclonal antibodyrecognizing Helicobacter pylori is selected therefrom, whereby thehybridoma of the invention can be obtained.

The above-mentioned immunogen is not particularly restricted but may beany one containing Helicobacter pylori catalase. Thus, for example,there may be mentioned, cultures obtainable by cultivating a strain ofHelicobacter pylori on an appropriate medium, cells in helical form,cells in coccoid form, disruption products, lysis products, orextraction products of such cells, and fractions thereof. The cellsmentioned above may be dead cells or viable cells.

The above catalase is not particularly restricted but may be, forexample, one deactivated, one having a partially destroyed stericstructure or one resulting from partial deletion. Preferred are,however, ones having four subunits, more preferably native ones.

The term “native enzyme” as used herein denotes one retaining theintrinsic structure found under approximately physiological conditionsand thus having all the subunits and the activity.

Among the immunogens mentioned above, a disruption product of coccoidcells is preferred. Since, in vitro, Helicobacter pylori morphologicallychanges from the ordinary helical form to a coccoid form, which cannotbe cultured, in unfavorable environmental conditions such as lowtemperature, nutritional deficiency, oxygen deficiency and the like, itis considered that it has been transformed into coccoid form, whichcannot be culturally isolated, in the digestive tract excreta as well.It is also considered that cells of Helicobacter pylori occur in adisrupted condition in the lower digestive tract.

The strain of Helicobacter pylori to be used as the above-mentionedimmunogen is not particularly restricted but includes, for example, thestrains ATCC 43504 and NCTC 11638, which are standard strains, and otherstrains isolated from infected persons. The genetic type of Helicobacterpylori to be used as the above immunogen is not particularly restricted.Thus, for example, it may have or may not have vacA or cagA and,further, vacA may have any of the sequences S1a, S1b and S2 and may haveany of the sequences m1 and m2.

The animal to be immunized for producing the hydridomas of the inventionis not particularly restricted but includes, for example, goats, sheep,guinea pigs, mice, rats, and rabbits. Among them, mice are preferred.

The above animals to be immunized can be immunized by any method knownin the art. In immunizing mice, for instance, there may be mentioned themethod comprising emulsifying 1 to 100 μg, preferably 50 to 100 μg, perdose, of the antigen for immunization in an equal volume (0.1 mL) ofphysiological saline and Freund's complete adjuvant or RIBI adjuvantsystem and administering the emulsion to the above animals to beimmunized subcutaneously at a dorsal or abdominal site orintraperitoneally 3 to 6 times at intervals of 2 to 3 weeks.

In the practice of the present invention, after immunization of theabove animals to be immunized, individuals high in antibody titer areselected, the spleen or lymph node of each of them is excised 3 to 5days after the final booster, and antibody-producing cells contained insuch tissue can be fused with myeloma cells by a cell fusion methodknown in the art in the presence of a fusion promoter.

The above fusion promoter is not particularly restricted but includes,for example, polyethylene glycol (hereinafter referred to as “PEG”),Sendai virus and so forth. PEG is preferred, however.

The above-mentioned myeloma cells are not particularly restricted butinclude, for example, mouse-derived cells, such as P3U1, NS-1 andP3×63.Ag8.653 cells; and rat-derived cells, such as AG1 and AG2 cells.

The above cell fusion method is not particularly restricted but mayinclude, for example, the method comprising mixing spleen cells withmyeloma cells in a ratio of 1:1 to 10:1, adding PEG having a molecularweight of 1,000 to 6,000 thereto to a concentration of 10 to 80% andincubating the mixture at 20 to 37° C., preferably 30 to 37° C., for 3to 10 minutes.

In the practice of the invention, the selection of hybridomas whichproduces a monoclonal antibody recognizing Helicobacter pylori can bemade, for example, by cultivating them on a selection medium, such asHAT medium, on which the hybridomas alone can grow, and measuring theantibody activity in each hybridoma culture supernatant by such a methodas enzyme-linked immunosorbent assay (ELISA). Furthermore, in thepresent invention, the establishment of a hybridoma, which produces amonoclonal antibody recognizing Helicobacter pylori, can be realized,for example, by subjecting the hybridoma which produces a monoclonalantibody recognizing Helicobacter pylori to repeated cloning by such amethod as limiting dilution.

As the hybridoma of the present invention, there may be mentioned, forexample, 21G2 (deposition number FERM BP-7336), 41A5 (deposition numberFERM BP-7337), and 82B9 (deposition number FERM BP-7338). Thesehybridomas have been deposited, as of Oct. 14, 1999, with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,Japan).

The method of preparing the monoclonal antibody of the invention inlarge quantities is not particularly restricted but may be, for example,the method comprising transplanting the hybridoma into the abdominalcavity of mice administered in advance with pristane, recovering theascitic fluid and obtaining the antibody from the same. The monoclonalantibody of the invention in ascitic fluid can be purified, for example,by a method known in the art using a protein A or protein G column, etc.

The monoclonal antibody of the invention recognizes catalase as anantigen.

The above catalase is not particularly restricted but may be any oneretaining the epitope. For example, it may be one deactivated, onehaving a partially destroyed steric structure, or one resulting frompartial deletion. One having four subunits is preferred, however, and anative one is more preferred. The above-mentioned catalase includesthose having a mutation or the like.

The strain of Helicobacter pylori, which produces the above-mentionedcatalase, is not particularly restricted.

The genetic type of Helicobacter pylori which produces the abovecatalase is not particularly restricted, either. For example, it mayhave or may not have vacA or cagA, and, further, vacA may have any ofthe sequences S1a, S1b and S2 and may have any of the sequences m1 andm2.

The morphology of the above Helicobacter pylori which produces catalaseis not particularly restricted but includes, for example, the helicalform of Helicobacter pylori and the coccoid form of Helicobacter pylori.

The class and subclass of the monoclonal antibody of the invention arenot particularly restricted but may be any of IgG₁, IgG₂, IgG₃, IgG₄,IgM, IgE, IgA₁, IgA₂ and IgD. The L chain is not particularlyrestricted, either, but may be the γ chain or κ chain.

The monoclonal antibody of the invention is not particularly restrictedbut may be any of those capable of specifically binding to Helicobacterpylori catalase. For example, it may be F(ab′)₂, Fab′ or Fab, which is adecomposition product of the monoclonal antibody of the invention, or achimera antibody.

The above hybridoma strains 21G2 (deposition number FERM BP-7336), 41A5(deposition number FERN BP-7337), and 82B9 (deposition number FERMBP-7338) produce respective monoclonal antibodies (hereinafterrespectively sometimes referred to also as “monoclonal antibody 21G2”,“monoclonal antibody 41A5”, and “monoclonal antibody 82B9”), whichrecognize native catalase of Helicobacter pylori.

The present inventors carried out antibody-immobilized affinitychromatography of a Helicobacter pylori cell disruption product usingthe monoclonal antibody 21G2, monoclonal antibody 41A5 and monoclonalantibody 82B9, respectively, upon which a protein having a molecularweight of 270 kDa as contained in the disruption product of Helicobacterpylori cells was detected. Upon SDS-polyacrylamide gel electrophoresisof this protein, a single band showing a molecular weight of 59 kDa wasdetected. Upon sequencing, the N-terminal amino acid sequence of thisprotein agreed with the amino acid sequence of Helicobacter pyloricatalase. It is known that Helicobacter pylori catalase has a molecularweight of 200 kDa and has a tetramer structure composed of 4 subunitseach having a molecular weight of 50 kDa (J. Gen. Microbiol. (1991),137, 57-61).

Therefore, base on the above results, the protein detected by themonoclonal antibody 21G2, monoclonal antibody 41A5 and monoclonalantibody 82B9 was identified as Helicobacter pylori catalase having foursubunits and it was revealed that each of the above-mentioned monoclonalantibodies recognizes the catalase having four subunits.

Catalase activity assay revealed that the catalase detected in the aboveantibody-immobilized affinity chromatography had activity and that thiscatalase was the so-called native enzyme.

None of the monoclonal antibody 21G2, monoclonal antibody 41A5 andmonoclonal antibody 82B9 reacted with the subunits of catalase resultingfrom dissociation.

The proportion of catalase to the whole protein in Helicobacter pyloricell is at most 0.5% on the weight basis as roughly calculated based onthe increase in specific activity in the process of purification ofcatalase as described in J. Gen. Microbiol. (1991), 137, 57-61.

In view of this point, it is a surprising and quite unexpected findingthat the monoclonal antibody 21G2, monoclonal antibody 41A5 andmonoclonal antibody 82B9 can recognize catalase having four subunits.

Catalase has been very well conserved among different species and it isknown that Helicobacter pylori catalase is highly homologous to otherbacterial catalase species (J. Bacteriol. (1996), 178 (23), 6960-6967).However, as shown in Example 3, the monoclonal antibody 21G2, monoclonalantibody 41A5 and monoclonal antibody 82B9 did not react at all withcatalase species derived from other bacterial species than Helicobacterpylori.

While it is also known that there exist various mutants of Helicobacterpylori (Molecular Microbiology (1996), 20, 833-842), the monoclonalantibody 21G2, monoclonal antibody 41A5 and monoclonal antibody 82B9surprisingly reacted well with all Helicobacter pylori strains, as shownin Example 3.

In view of the fact that the monoclonal antibodies of the presentinvention react well with various mutants of Helicobacter pylori, theepitope which is recognized by the monoclonal antibodies of theinvention seems to be a site well conserved among catalase specieswithin mutants.

As will be mentioned below in detail, the monoclonal antibodies of theinvention make it possible to make judgment about the occurrence ornonoccurrence of Helicobacter pylori infection with very high accuracyusing feces as specimens.

Feces specimens were subjected to immunological assay using themonoclonal antibody 21G2, monoclonal antibody 41A5 and monoclonalantibody 82B9. The above monoclonal antibodies each reacted only withthe feces of subjects infected with Helicobacter pylori. By this, it wasrevealed that Helicobacter pylori catalase having four subunits ispresent in the feces of subjects infected with Helicobacter pylori. Ithas been quite unknown in the art that the Helicobacter pylori catalaseoccurring in feces is not dissociated into each subunit but retains thefour subunits. This is quite surprising in view of the fact thatproteins are generally digested by proteases in the digestive tract.

A column was prepared by immobilizing the monoclonal antibody 21G2thereon and affinity chromatography was carried out using, as samplesfeces from persons infected with Helicobacter pylori, and the eluatefractions were subjected to ELISA and catalase activity assay. Thecatalase activity was in agreement with the antigenicity.

In view of the foregoing, it was revealed that the feces of personsinfected with Helicobacter pylori contain native catalase having foursubunits and catalase activity. It has been quite unexpected thatHelicobacter pylori catalase is not digested in the digestive tract butis excreted in the form of native enzyme having activity and thus occursin feces.

In Japanese Kokai Publication Hei-09-322772 and Infect. Immun. (1997),65, 4668-4674, there are disclosed monoclonal antibodies which reactwith a protein corresponding to each disentangled subunit obtainable bydenaturation and dissociation of Helicobacter pylori catalase bySDS-PAGE. However, it is not clear whether these monoclonal antibodiesreact with native catalase retaining the steric structure and havingactivity or whether they can detect Helicobacter pylori in excretaspecimens, such as feces.

On the contrary, the monoclonal antibody of the present invention makesit possible to recognize the occurrence or nonoccurrence of Helicobacterpylori infection using digestive tract excreta, such as feces, which canbe collected with ease, as specimens.

The field of application of the monoclonal antibody of he presentinvention is not particularly restricted but can be used, inimmunological assays, for making judgment about Helicobacter pyloriinfection.

The immunological assay method of the present invention is carried outusing at least one monoclonal antibody species according to theinvention, and the method can be carried out using only one species.

Generally, as the monoclonal antibody used in the immunological assaymethod, monoclonal antibodies, which are superior in sensitivity, isconsidered as preferred because of higher specificity and lowerbackground noise compared with polyclonal antibodies, which are low inspecificity and high in background noise.

It is known, however, that there are various mutants of Helicobacterpylori. Therefore, it has been considered that it would be verydifficult to detect the infection or occurrence using a singlemonoclonal antibody. As described in Japanese Kokai PublicationHei-10-10128, there is also an opinion that polyclonal antibodies, whichcan react with a variety of antigens, are more suited for the detectionof infection with or occurrence of a bacterial species having a varietyof antigens. Further, a method of detecting Helicobacter pylori using aplurality of monoclonal antibodies has been disclosed (WO 00/26671).However, any method has not yet been developed for detectingHelicobacter pylori using a single monoclonal antibody.

On the contrary, it has surprisingly been found that the monoclonalantibody of the present invention can detect Helicobacter pylori withvery high accuracy using a single antibody species.

The monoclonal antibodies described in WO 00/26671 recognize urease,heat shock proteins, alkaline hydroperoxidase reductase, 20 kDa protein(3-dehydrokinase type 2), 16.9 kDa protein (neutrophile activatingprotein) and 33.8 kDa protein (fructose-bisphosphatase aldolase) asantigens. Catalase is not mentioned at all. The assay method describedin WO 00/26671 is not satisfactory in detection sensitivity.

As the immunological assay method of the present invention, there may bementioned, for example, enzyme immunoassay (EIA), enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescenceimmunoassay (FIA), western blotting, immunochromatography and liketechniques. The various immunological assay methods mentioned above canbe used in assaying the target antigens or antibodies by using anantigen or antibody labeled with a labeling agent in the manner ofcompetitive or sandwich method, etc.

Among the above-mentioned various immunological assay methods, ELISA andimmunochromatography techniques are preferred.

The above competitive method is based, for example, on thequantitatively competitive binding reaction of Helicobacter pyloricatalase in a test specimen and a known amount of labeled Helicobacterpylori catalase to the monoclonal antibody of the present invention. Inthe competitive method specifically mentioned above, a predeterminedamount of the antibody immobilized on a carrier and a predeterminedamount of Helicobacter pylori catalase labeled with a labeling agent areadded to a specimen solution containing Helicobacter pylori catalase.Then, the activity of the labeling agent retained on the carrier or ofthe labeling agent not retained on the carrier is determined. In thiscase, it is preferable to add the antibody and labeled antigen almostsimultaneously.

The above-mentioned sandwich method comprises, for example, sandwichingHelicobacter pylori catalase in a specimen between the immobilizedmonoclonal antibody of the invention and the monoclonal antibody of theinvention labeled with a labeling agent, and then adding a substrate orthe like against the labeling agent such as an enzyme, to cause colordevelopment or the like, and thereby detect Helicobacter pylori catalasein the specimen.

As the above labeling agent, there may be mentioned radioisotopes(hereinafter abbreviated as “RI”) such as ¹²⁵I, enzymes, enzymesubstrates, luminescent substances, fluorescent substances, biotin, andcolored substances. In binding these labeling agents to the antigen orantibody, the maleimide method [J. Biochem. (1976), 79, 233], activatedbiotin method [J. Am. Chem. Soc. (1978), 100, 3585] or hydrophobic bondmethod, for instance, can be used.

As the enzyme mentioned above, there may be mentioned, for example,peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase.As for the substrate to be used on that occasion, one suitable for theenzyme employed may be selected and there may be mentioned, for example,ABTS, luminol-H₂O₂, o-phenylenediamine-H₂O₂ (against peroxidase),p-nitrophenyl phosphate, methylumbelliferyl phosphate,3-(2′-spiroadamantan)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane(against alkaline phosphatase), p-nitrophenyl-β-D-galactose, andmethylumbelliferyl-β-D-galactose (against β-galactosidase).

The above assay can be carried out by allowing the reaction to proceedat 4 to 40° C. for 1 minute to 18 hours and then measuring the resultingdeveloped color or amount of fluorescence, luminescence, or coloration.Alternatively, the so-called rate assay may be employed which is carriedout while incubating in the range of 4 to 40° C.

The radiolabeling to the above antigen or antibody can easily be carriedout using the Bolton-Hunter reagent, which is commercially available.For example, it can be carried out by adding the Bolton-Hunter reagentto a solution prepared by dissolving an antigen or antibody in 0.1 Maqueous solution of sodium hydrogen carbonate and, after the lapse of 1to 2 hours, removing the unreacted portion of the Bolton-Hunter reagentusing a G-25 desalting column or the like.

In addition, the radiolabeling with ¹²⁵I can be easily carried out byemploying the chloramine T method, the iodogen method or the like.

As the above luminescent substance, there may be mentioned, for example,isoluminol and acridine esters and, as the above fluorescent substance,there may be mentioned, for example, fluorescein and rhodamine. In thiscase, the labeling can be carried out with ease by employing theactivated ester method or the isocyanate method (“Enzyme immunoassaytechniques”, published in 1987 by Igaku Shoin). As the above coloredsubstance, there may be mentioned, for example, colored latex particlesand colloidal gold.

For carrying out the immunological assay method of the present inventionby the above-mentioned competitive method, a specimen containing anunknown amount of Helicobacter pylori catalase, for instance, is addedto a solid phase to which the monoclonal antibody of the presentinvention is bound physically or chemically by known means, and thereaction is allowed to proceed. Simultaneously, a predetermined amountof Helicobacter pylori catalase labeled with a labeling agent is addedand the reaction is allowed to proceed.

In carrying out the immunological assay method of the present inventionby the above-mentioned sandwich method, a specimen containing an unknownamount of Helicobacter pylori catalase, for instance, is added to asolid phase to which the monoclonal antibody of the present invention isbound physically or chemically by known means, and the reaction isallowed to proceed. Thereafter, the monoclonal antibody of the inventionlabeled with a labeling agent is added and the reaction is allowed toproceed.

Then, in both cases, the solid phase is thoroughly washed, if necessary,and the activity of the labeling agent bound to the solid phase ismeasured. When the above labeling agent is R1, the measurement iscarried out using a well counter or a liquid scintillation counter. Whenthe labeling agent is an enzyme, the substrate is added and, afterstanding, the enzyme activity is measured by colorimetry or fluorometry.When the labeling agent is a fluorescent substance, luminescentsubstance or colored substance, the measurement can be made respectivelyby a method known in the art.

The immunological assay method of the present invention uses themonoclonal antibody of the present invention and therefore hasoutstanding characteristics, namely the epitope specifically occurringin Helicobacter pylon catalase can be recognized without erroneouslydetecting other substances as a result of a cross reaction and thusmeasurements can be carried out with very high specificity.

For carrying out the immunological assay method of the presentinvention, the diagnosis kit of the present invention can be used. Thediagnosis kit of the invention comprises at least one monoclonalantibody species of the invention and may comprise only one monoclonalantibody species. The monoclonal antibody of the invention, which is tobe used in the diagnosis kit of the invention, is not particularlyrestricted but may be any of those recognizing Helicobacter pyloricatalase, and may be the decomposition products of the monoclonalantibody of the invention such as F(ab′)₂, Fab′, Fab and the like.

The diagnosis kit of the invention can immunologically detect theoccurrence of Helicobacter pylori cells or of Helicobacter pyloricatalase and therefore makes judgment about Helicobacter pyloriinfection.

In the diagnosis kit of the invention, the monoclonal antibody of theinvention may be immobilized on a solid phase in advance, and themonoclonal antibody of the invention may be labeled with theabove-mentioned labeling agent in advance.

The solid phase to be used in the diagnosis kit of the invention is notparticularly restricted but includes, for example, polymers such aspolystyrene, glass beads, magnetic particles, microplates, filter paperfor immunochromatography, glass filters and other insoluble carriers.

The diagnosis kit of the invention may further comprise otherconstituents.

The other constituents mentioned above are not particularly restrictedbut include, for example, enzymes for labeling, substrates therefor,radioisotopes, luminescent substances, fluorescent substances, coloredsubstances, buffer solutions, and plates, and those mentionedhereinabove can be used as these.

Although the form of the diagnosis kit of the invention is notparticularly restricted, but integrated type diagnosis kit comprisingthe constituents of the diagnosis kit of the invention together ispreferred in order to carry out the diagnosis in a rapid, simple andeasy manner.

The above integrated type diagnosis kit is not particularly restrictedbut may be of the cassette type, cartridge type, or the like.

As an embodiment of the above-mentioned cassette type, there may bementioned, for example, an embodiment which comprises, using aimmunochromatography technique, a reaction cassette, a membranecontained therein, the monoclonal antibody of the invention immobilizedon said membrane at one end thereof (downstream side), a developingsolution provided on the membrane at the opposite end thereof (upstreamside), a pad containing a substrate against the above-mentioned labelingagent and disposed in the vicinity of the developing solution on theside downstream side thereof, and a pad containing the monoclonalantibody of the invention labeled with the above-mentioned labelingagent and disposed in the middle portion of the membrane.

In using the diagnosis kit having the embodiment of the cassette typementioned above by way of example, the specimen is applied onto the padcontaining the monoclonal antibody of the invention labeled with theabove labeling agent and, after allowing the formation of the bondingproduct between Helicobacter pylori catalase contained in the specimenand the monoclonal antibody of the invention labeled with the labelingagent, the above-mentioned developing solution is allowed to develop tothereby transfer the bonding product formed to the site in which themonoclonal antibody of the invention is immobilized, where the complexof Helicobacter pylori catalase, the monoclonal antibody of theinvention labeled with the labeling agent and the immobilized monoclonalantibody of the invention is formed. Then, the above-mentioned labelingagent reacts with the above-mentioned substrate to develop a color orthe like. This developed color or the like is measured, whereby judgmentabout Helicobacter pylori infection can be made.

In cases where an embodiment comprising diluting the specimen using asufficient amount of a developing solution and dropping the resultingsolution onto the membrane is empolyed, it is not necessary to providethe developing solution on the membrane in advance. When a coloredsubstance such as colored latex particles is used as the above-mentionedlabeling agent, no substrate is required and whether the above-mentionedcomplex is formed or not at the site in which the monoclonal antibody ofthe invention is immobilized can be judged based on the coloration dueto the colored substance.

As an embodiment of the above-mentioned cartridge type for carrying outthe reaction by the above-mentioned competitive method, for instance,there may be mentioned a cartridge or the like having a plurality ofwells, which are integrally formed, and comprising (a) a well containingthe monoclonal antibody of the invention, (b) a well containing a liquidreagent (e.g. buffer solution) containing Helicobacter pylori labeledwith the above-mentioned labeling agent, and (c) a well containing aliquid reagent (e.g. buffer solution) containing a substrate against theabove-mentioned labeling agent and, for carrying out the reaction by thesandwich method, there may be mentioned a cartridge or the like having aplurality of wells, which are integrally formed, and comprising (a) awell containing a insoluble carrier on which the monoclonal antibody ofthe invention is immobilized, (b) a well containing a liquid reagent(e.g. buffer solution) containing the monoclonal antibody of theinvention labeled with the above-mentioned labeling agent, and (c) awell containing a liquid reagent (e.g. buffer solution) containing asubstrate against the above-mentioned labeling agent.

In using the cartridge type diagnosis kit of the present inventionmentioned above by way of example, the reaction and assay can generallybe carried out in the same manner as in carrying out the competitive orsandwich method.

The diagnosis kit of the invention may be one for carrying out theabove-mentioned competitive method or the above-mentioned sandwichmethod. One for carrying out the sandwich method is preferred, however.The above sandwich method has advantages, namely it is high insensitivity, requires a short reaction time, and is superior inaccuracy. By using immunochromatography technique as the above sandwichmethod, it becomes possible to produce a kit which renders the testprocedure easy and simple and with which the judgment of the results caneasily be done by visual observation. When the above sandwich method isused in the manner of ELISA technique, the enzyme reaction product isformed depending on the amount of the assay target substance andtherefore a system by which the color development or the like in thecase of positive Helicobacter pylori infection can be confirmed byvisual observation can easily be established.

The specimen to be used with the diagnosis kit of the present inventionis not particularly restricted but includes, for example, gastriccontents, gastric washings, and digestive tract excreta. Digestive tractexcreta, such as feces, are preferred, however, since they can easily becollected without imposing any burden on subjects.

The diagnosis kit of the present invention may be used for examining forthe occurrence or nonoccurrence of the infection prior to bacterialeradication treatment and/or for making judgment about the success orfailure thereof after bacterial eradication treatment.

The diagnosis kit of the present invention has a high level of detectionsensitivity and produces no false negative or false positive problems,without any difference among lots since it uses a monoclonal antibody.

In accordance with the present invention, the influences of othersubstances occurring in the specimen can be eliminated by using themonoclonal antibody recognizing Helicobacter pylori catalase as anantigen, so that the occurrence of Helicobacter pylori can be detectedwith very high sensitivity and specificity. Since the hybridoma whichproduces a monoclonal antibody against Helicobacter pylori catalase havebeen established, it is now possible to produce the same monoclonalantibody almost semi-permanently. The diagnosis kit in which themonoclonal antibody of the present invention is used can use digestivetract excreta as specimens, so that Helicobacter pylori infection can bedetected in a simple and easy manner and efficiently without causing anypain on subjects. Further, in cases where only one monoclonal antibodyspecies is used, the diagnosis kit of the invention in which themonoclonal antibody of the present invention is used can stably attainvery high accuracy without any difference among lots, hence can detectHelicobacter pylori infection always specifically and with greataccuracy. The above diagnosis kit is easy in carrying out the assay andis very useful at the site of medical care.

BEST MODES FOR CARRYING OUT THE INVENTION

The following examples and test examples illustrate the presentinvention in further detail. They are, however, by no means limitativeof the scope of the present invention.

EXAMPLE 1

[Monoclonal Antibody Production]

(1) Preparation of a Helicobacter pylori Coccoid Cell Suspension(Immunogen)

Brain heart infusion agar medium (product of Difco) supplemented with 5%equine defibrinated blood was streaked with Helicobacter pylori (ATCC43504) in series. The plate was incubated at 37° C. for 3 to 4 days in amicroaerobic environment and then further incubated at 37° C. for 7 daysin an anaerobic environment to cause the cells to become coccoid. Eachcolony obtained was scraped off with a platinum loop or the like andsuspended in phosphate-buffered physiological saline (PBS). Helicobacterpylori cells were collected by 10 minutes of centrifugation at 10,000×gat 4° C. and suspended in 0.5 % formalin, and the suspension was allowedto stand at 4° C. for 4 days for inactivation. Thereafter, the cellswere washed by three repetitions of the procedure comprising suspendingin PBS followed by 10 minutes of centrifugation at 10,000×g at 4° C. andthen suspended in PBS.

(2) Preparation of a Disruption Product of Helicobacter pylori CoccoidCells (Immunogen)

The cell suspension obtained in (1) was disrupted by 10 minutes ofultrasonication using an ultrasonic disrupter (product of Seiko DenshiKogyo, Model 7250) under output 3.50% duty cycle conditions.

(3) Immunization and Cell Fusion

Equal amounts of the immunogen prepared as described under (1) or (2)(Helicobacter pylori coccoid cell suspension or the disruption productof Helicobacter pylori cells) and Freund's complete adjuvant (product ofCalbiochem) were mixed up to give an oil emulsion. This emulsion wassubcutaneously administered to BALB/cA mice (products of CLEA Japan,6-week-old, males) at a dorsal site at a dose of 0.2 mL. Booster wascarried out 7 days and 14 days after the first immunization and,further, 3 days prior to cell fusion, 0.2 mL of the above immunogen wasintraperitoneally administered. Three days after the final booster, thespleen cells were excised from each mouse and mixed with myeloma cells(P3×63.Ag8.653 strain, RCB 0146, Riken Gene Bank) in a ratio of 10:1 andfused together using 50% polyethylene glycol 4000. Hybridomas werecultured selectively on HAT medium (product of Gibco).

(4) Hybridoma Selection

On the 12th day after cell fusion, the antibody activity in each culturesupernatant was measured by ELISA. The culture supernatant of fusioncells, 200-μl each, was added to wells of a 96-well ELISA plate (productof Coaster) with 10 μg/mL of the immunogen immobilized thereon and,after allowing the reaction to proceed at 37° C. for 1 hour and washingwith 0.05% Tween 20-containing PBS (washing solution), 200 μL ofperoxidase-labeled anti-mouse IgG (product of Cappel, 1:20,000) wasadded to each well. After allowing the reaction to proceed at 37° C. for1 hour, the plate was washed with the above washing solution.

Thereafter, 200 μL of a substrate solution (0.1 M o-phenylenediamine and0.012% aqueous hydrogen peroxide) was added to each well and thereaction was allowed to proceed at room temperature for 15 minutes.Thereafter, the enzyme reaction was terminated by adding 50 μL of 3.5 Nsulfuric acid to each well, and the absorbance at 492 nm was measured.Those hybridomas clones which produced an antibody reactive with theabove immunogen and gave an absorbance not lower than 0.15 wereselected. Each clone was subjected to two repetitions of cloning by thelimiting dilution method. Hybridomas after cloning were transplantedinto BALB/cA mice and, as a result, 32 hybridoma clones were found toproduce respective monoclonal antibodies that can be recovered asascitic fluids.

EXAMPLE 2

[Selection of Monoclonal Antibodies Specifically RecognizingHelicobacter pylori in Digestive Tract Excreta by Sandwich ELISA]

(1) Preparation of Monoclonal Antibody-Immobilized Plates

Each 1-mL portion of the ascetic fluids of the 32 clones was dilutedtwo-fold with PBS, 2 mL of saturated ammonium sulfate was addeddropwise, and the mixture was allowed to stand at 4° C. for 4 hours.Then, the mixture was centrifuged at 3000 rpm for 20 minutes, and thesediment was suspended in 2 mL of PBS and dialyzed. These monoclonalantibodies were immobilized on 96-well ELISA plates in the followingmanner. Thus, each monoclonal antibody was diluted to 5 μg/mL and 0.2 mLof the dilution was added to each well of 96-well ELISA plates. Afterovernight standing at 4° C., the plates were washed with PBS, then 0.25mL of 1% skim milk-PBS was added to each well and the plates wereallowed to stand at 4° C. for 1 hour for blocking. Thereafter, theplates were washed with the above washing solution.

(2) Preparation of Biotin-Labeled Monoclonal Antibodies

Each 3-mg portion of the 32-clone monoclonal antibodies prepared in (1)was mixed with 10 mg of biotinyl N-hydroxysuccinimide ester (product ofZymed) and the reaction was carried out in 0.1 M sodium hydrogencarbonate (pH 8) with stirring at room temperature for 3 hours. Thereaction mixture was dialyzed overnight against 5 L of PBS at 4° C. togive the biotin-labeled monoclonal antibody.

(3) Selection of Monoclonal Antibodies Specifically recognizingHelicobacter pylori in digestive tract excreta

Feces specimens, 250 mg each, from one person judged as Helicobacterpylori positive and one person judged as negative by the urea breathtest were each suspended in 0.5 mL of 0.1% skim milk-PBS, each mixturewas centrifuged at 3,000 rpm for 10 minutes, and the supernatant thusseparated was used as a fecal extract. 0.2 mL of each fecal extract wasadded to each well of the monoclonal antibody-immobilized plate preparedas described in (1). After 1 hour of standing at 37° C., the plate waswashed with 5 portions of the above washing solution, and 0.2 mL of eachbiotin-labeled monoclonal antibody prepared in (2) was added. After 1hour of standing at 37° C., the plate was washed with she above washingsolution, and 0.2 mL of peroxidase-labeled avidin (product of Zymed) wasadded. After 1 hour of standing at 37° C., the plate was washed with theabove washing solution, 0.2 mL of a substrate solution (0.1 Mo-phenylenediamine and 0.012% aqueous hydrogen peroxide) was added toeach well, and the reaction was allowed to proceed at room temperaturefor 10 minutes. Thereafter, the enzyme reaction was terminated by adding50 μL of 3.5 N sulfuric acid, and the absorbance was measured at 492 nm.As shown in Table 1, sandwich ELISA in which three monoclonalantibodies, 21G2, 41A5 and 82B9, used singly or in combination, werefound to show high reactivity against the fecal specimen of the personinfected with Helicobacter pylori.

TABLE 1 Combination of monoclonal antibodies Absorbance ImmobilizedLabel Positive subject Negative subject 21G2 21G2 3.3 0.043 21G241A5 >3.5 0.075 21G2 82B9 >3.5 0.067 41A5 41A5 3.3 0.10 41A5 21G2 >3.50.080 41A5 82B9 >3.5 0.035 82B9 82B9 >3.5 0.071 82B9 21G2 3.3 0.066 82B941A5 >3.5 0.064

The hybridomas producing the respective monoclonal antibodies have beendeposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology under thedesignations hybridoma 21G2 (FERM BP-7336), hybridoma 41A5 (FERMBP-7337) and hybridoma 82B9 (FERM BP-7338). The immunoglobulin subclassof each monoclonal antibody was checked using an immunoglobulin typingkit mouse (product of Wako Pure Chemical Industries), and as a result,all the three clones were found to belong to IgG₁ with κ type L chains.

EXAMPLE 3

[Reactivity Comparison Among Strains and Reactivity with Other BacterialSpecies]

(1) Preparation of Helicobacter pylori Cell Suspensions

Brain heart infusion agar medium supplemented with 5% equinedefibrinated blood was streaked with each of Helicobacter pylori strains(ATCC 43504; Tokai University Hospital clinical isolates No. 130 and No.112; Hyogo Medical College clinical isolates No. 526, No. 4484, No.5017, No. 5025, No. 5049, No. 5142, No. 5287, No. 5308, No. 5314 and No.5330) in series. The plates were incubated at 37° C. under microaerobicconditions for 3 to 4 days to give colonies of helical cells, or furtherallowed to stand a: 37° C. for 7 days in an anaerobic environment togive colonies of coccoid cells. Each colony was scraped off with aplatinum loop or the like and suspended in PBS. Cells were collected by10 minutes of centrifugation at 10,000×g at 4° C. and suspended in 0.5%formalin, and the suspension was allowed to stand at 4° C. for 4 daysfor inactivation. Thereafter, the cells were washed by three repetitionsof the procedure comprising suspending in PBS, followed by 10 minutes ofcentrifugation at 10,000×g at 4° C., and then again suspended in PBS.Thus were obtained Helicobacter pylori helical cell suspensions andcoccoid cell suspensions.

(2) Preparation of Human Enterobacterial and Campylobacter Jejuni CellSuspensions

Typical bacterial species isolable from human feces, namely twoenterobacterial species Bacteroides vulgatus and Escherichia coli, andthe helical bacterial species Campylobacter jejuni were cultured in themanner mentioned below and, from the colonies obtained, each cellsuspension was prepared in the same manner as mentioned in (1). Thus,Bacteroides vulgatus (IFO 14291) was anaerobically cultured on BL agarmedium (product of Nissui Pharmaceutical) supplemented with 5% equinedefibrinated blood at 37° C. for 2 days. Escherichia coli (ATCC 25922)was aerobically cultured on brain heart infusion agar medium at 37° C.for 1 day. Campylobacter jejuni (80068, Tokai University Hospitalclinical isolate) was microaerobically cultured on brain heart infusionagar medium supplemented with 5% equine defibrinated blood at 37° C. for2 days.

(3) Preparation of Cell Suspensions of Other Helicobacter SpeciesHelicobacter felis (ATCC 49179) and Helicobacter hepaticus (ATCC 51448)were cultured on brain heart infusion agar medium supplemented with 5%equine defibrinated blood in the same manner as in (1) and, from thethus-obtained colonies of helical cells, helical cell suspensions wereobtained in the same manner as in (1).

(4) Preparation of Cell Disruption Products

Each cell suspension obtained in (1), (2) or (3) was subjected toultrasonication for cell disruption using an ultrasonic disrupter(product of Seiko Denshi Kogyo, Model 7250) under output 3.50% dutycycle conditions.

(5) Sandwich ELISA

Each cell disruption product obtained in (4) (0.2 mL; proteinconcentration 10 μg/mL) was subjected to sandwich ELISA by the method ofExample 2 (3) (monoclonal antibody 41A5-immobilized plate:biotin-labeled monoclonal antibody 82B9, monoclonal antibody21G2-immobilized plate: biotin-labeled monoclonal antibody 41A5,monoclonal antibody 21G2-immobilized plate: biotin-labeled monoclonalantibody 82B9, monoclonal antibody 21G2-immbolized plate: biotin-labeledmonoclonal antibody 21G2, and Meridian's product HpSA). The results areshown in Table 2.

TABLE 2 Absorbance Anti- Immobilized 21G2 41A5 21G2 21G2 HpSA bodymonoclonal antibody Labelled 41A5 82B9 82B9 21G2 monoclonal antibodyAnti- Helicobacter >3.5 >3.5 >3.5 >3.5 >3.5 gen pylori ATCC 43504helical cells Helicobacter >3.5 >3.5 >3.5 — — pylori ATCC 43504spherical cells Helicobacter >3.5 >3.5 >3.5 — — pylori No. 130 helicalcells Helicobacter >3.5 >3.5 >3.5 — — pylori No. 130 spherical cellsHelicobacter >3.5 >3.5 >3.5 — — pylori No. 112 helical cellsHelicobacter >3.5 >3.5 >3.5 — — pylori No. 112 spherical cellsHelicobacter — — — >3.5 — pylori No. 526 helical cells Helicobacter —— >3.5 — pylori No. 4484 helical cells Helicobacter — — — >3.5 — pyloriNo. 5017 helical cells Helicobacter — — — >3.5 — pylori No. 5025 helicalcells Helicobacter — — — >3.5 — pylori No. 5049 helical cellsHelicobacter — — — >3.5 — pylori No. 5142 helical cells Helicobacter — —— >3.5 — pylori No. 5287 helical cells Helicobacter — — — >3.5 — pyloriNo. 5308 helical cells Helicobacter — — — >3.5 — pylori No. 5314 helicalcells Helicobacter — — — >3.5 — pylori No. 5330 helical cellsBacteroides 0.066 0.066 0.058 — — vulgatus IFO 14291 Escherichia coli0.056 0.055 0.050 — — ATCC 25922 Campylobacter 0.079 0.057 0.062 — —jejuni 80068 Helicobacter felis 0.066 0.051 0.055 0.014 1.63 ATCC 49179helical cells Helicobacter 0.059 0.049 0.061 0.016 0.13 hepaticus ATCC51448 helical cells

As shown in Table 2, the monoclonal antibodies 21G2, 41A5 and 82B9 werefound to show high reactivity against helical cells and coccoid cells ofeach of Helicobacter pylori strains. On the other hand, they did notreact at all with Bacteroides vulgatus, Escherichia coli, Campylobacterjejuni, Helicobacter felis or Helicobacter hepaticus. On the contrary,Meridian's HpSA showed reactivity against Helicobacter fells andHelicobacter hepaticus as well.

EXAMPLE 4

[Detection of Helicobacter pylori in Fecal Specimens by Sandwich ELISA(1)]

Feces specimens (each 250 mg) from three persons judged as Helicobacterpylori positive and three persons judged as negative by the urea breathtest were each suspended in 0.5 mL of 0.1% skim milk-PBS, and eachsuspension was tested in the same manner as in Example 2 (3). The samefecal specimens were also subjected to the test using Meridian's HpSAELISA according to the procedure indicated, and the absorbance values at450 nm were measured. The respective absorbance values and urea breathtest results are shown in Table 3.

TABLE 3 Combination of monoclonal antibodies Absorbance (urea-expiredair test alone, per mil) Immobilized Label No. 1 No. 2 No. 3 No. 4 No. 5No. 6 21G2 21G2 0.041 0.043 0.043 3.3 2.8 2.1 21G2 41A5 0.092 0.0790.075 >3.5 >3.5 2.4 21G2 82B9 0.071 0.052 0.067 >3.5 >3.5 3.2 41A5 41A50.17  0.17  0.10  3.3 >3.5 0.84 41A5 21G2 0.077 0.073 0.080 >3.5 >3.51.8 41A5 82B9 0.029 0.053 0.035 >3.5 >3.5 2.2 82B9 82B9 0.12  0.0860.071 >3.5 >3.5 2.3 82B9 21G2 0.063 0.082 0.066 3.3 >3.5 1.1 82B9 41A50.079 0.077 0.064 >3.5 >3.5 >3.5 HpSA 0.077 0.028 0.025 1.8 2.5 1.8Urea-expired air test (1.0) (0.6) (0.9) (17) (26) (17)

The fecal specimens provided by the subjects (No. 4, 5, 6) whose ureabreath test results were positive were found to show markedly higherabsorbance values as compared with the negative specimens (No. 1, 2, 3).

EXAMPLE 5

[Detection of Helicobacter pylori in Fecal Specimens by Sandwich ELISA(2)]

(1) Monoclonal Antibody Preparation

The ascitic fluid (5 mL) recovered from a BALB/cA mouse transplantedwith the hybridoma 21G2 was diluted two-fold with PBS, 10 mL ofsaturated ammonium sulfate was added dropwise, and the mixture wasallowed to stand at 4° C. for 4 hours. The mixture was then centrifugedat 3,000 rpm for 20 minutes, the sediment was dissolved in 10 mL of PBS,and the solution was dialyzed against PBS.

(2) Preparation of Monoclonal Antibody-Immobilized Plates

The monoclonal antibody 21G2 solution prepared in (1) was immobilized on96-well ELISA plates in the following manner. The antibody was dilutedto a concentration of 5 μg/mL with PBS and the dilution was distributedin 0.2-mL portions into each well of the 96-well ELISA plates and, afterovernight standing at 4° C., the plates were washed with PBS.

(3) Preparation of a Peroxidase-Labeled Monoclonal Antibody

A peroxidase-labeled monoclonal antibody was prepared using themaleimide method (Eiji Ishikawa: “Experiments in Biological Chemistry”,vol. 27, Enzyme labeling techniques, p. 51, published 199 by GakkaiShuppan Center). The monoclonal antibody (5 mg) prepared in (1) wasmixed with 0.6 mg of S-acetylmercaptosuccinic anhydride (product orAldrich) and the reaction was allowed to proceed in 0.5mL of 0.1 Mphosphate buffer (pH 6.5) at 30° C. for 30 minutes. To the reactionmixture were added 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Trishydrochloride buffer (pH 7.0) and 0.1 mL of 1 M hydroxylamine (pH 7.0),and the mixture was allowed to stand at 30° C. for 5 minutes and thencentrifuged at 10,000 rpm for 5 minutes to give a supernatant. Thesupernatant was subjected to ultrafiltration using Centricon 30 (productof Amicon) to thereby remove the reagents, and the solvent was replacedwith 1 mL of 5 mM EDTA-0.1 M phosphate buffer (pH 6.5), whereby thethiol group-introduced monoclonal antibody.

Peroxidase (5 mg, horseradish, product of Toyobo) was mixed with 1 mg ofN-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (productof ICN Biomedicals), and the reaction was allowed to proceed in 0.5 mLof 0.1 M phosphate buffer (pH 7.0) at 30° C. for 1 hour. The reactionmixture was centrifuged at 10,000 rpm for 5 minutes to give asupernatant. The supernatant was subjected to ultrafiltration usingCentricon 30 to remove the reagents, the solvent was then replaced with1 mL of 0.1 M phosphate buffer (pH 7.0), whereby the maleimidegroup-introduced peroxidase was obtained.

The above thiol group-introduced monoclonal antibody and maleimidegroup-introduced peroxidase were mixed together in a molar ratio of 1:5,and the reaction was allowed to proceed at room temperature for 30minutes. The reaction mixture was applied to a gel filtration column(Sephacryl-S 300 HR column; diameter 26×length 870 mm, 0.1 M phosphatebuffer (pH 6.5)) and a fraction containing the peroxidase-labeledmonoclonal antibody was collected.

(4) Detection of Helicobacter pylori in Fecal Specimens by SandwichELISA

Feces specimens (each 250 mg) from ten persons judged as Helicobacterpylori positive and ten persons judged as negative by the urea breathtest were each suspended in 0.5 mL of 0.1% skim milk-PBS, each mixturewas centrifuged at 3,000 rpm for 10 minutes, and the supernatant thusobtained was used as a fecal extract. 50 μL of each fecal extract and 50μL of the peroxidase-labeled monoclonal antibody prepared in (3) wereadded to each well of the above-mentioned monoclonalantibody-immobilized plate. After 1 hour of standing at 25° C., theplate was washed with 5 portions of a washing solution (0.05% Tween20-PBS), 0.1 mL of a substrate solution (tetramethylbenzidine+hydrogenperoxide, product of BioFX) was added to each well, and the reaction wasallowed to proceed at room temperature for 10 minutes. Thereafter, theenzyme reaction was terminated by adding 50 μL of 1 N sulfuric acid toeach well, and the absorbance (450 nm-630 nm) was measured. The samefecal specimens were tested using HpSA and the absorbance (450 nm-630nm) was measured. The results are shown in Table 4.

TABLE 4 Urea-expired 21G2 sandwich Speci- air test ELISA HpSA menJudgment^(*1) Judgment^(*2) Judgment^(*3) No. (per mil) (Absorbance)(Absorbance)  1 + (17) + (1.89) + (1.30)  2 + (11) + (1.19) + (1.57)  3− (40) + (1.52) + (1.64)  4 + (40) + (1.24) + (2.13)  5 + (25) +(0.46) + (2.11)  6 + (34) + (0.79) + (1.37)  7 + (49) + (1.38) + (1.60) 8 + (50) + (3.13) + (2.18)  9 + (19) + (0.36) + (0.76) 10 + (40) +(2.43) + (1.97) 11 − − (0.05) − (0.01) 12 − − (0.02) − (<0.01) 13 − −(0.04) − (<0.01) 14 − − (0.09) − (0.01) 15 − − (0.02) − (<0.01) 16 − −(0.03) + (0.27) 17 − − (0.02) − (<0.01) 18 − − (0.04) − (0.01) 19 − −(0.05) + (0.23) 20 − − (0.01) − (0.02) ^(*1): + Positive (>5 per mil), −Negative ^(*2): + Positive (>0.1), − Negative ^(*3): + Positive (>0.1),− Negative

As shown in Table 4, the sandwich ELISA using the monoclonal antibody21G2 singly showed high reactivity with the fecal specimens from personsinfected with Helicobacter pylori in precise agree-ent with the judgmentresults of the urea breath test. On the other hand, HpSA gavedisagreements (false positives) with two specimens (specimen Nos. 16 and19).

EXAMPLE 6

[Detection of Helicobacter pylori in Fecal Specimens byImmunochromatography]

(1) Preparation of Antibody-Immobilizing Supports

For preparing antibody-immobilizing supports with the anti-Helicobacterpylori monoclonal antibody and anti-rabbit IgG antibody immobilizedthereon in series, a nitrocellulose sheet (product of Whatman) was cutto 5 mm×20 mm, and a solution of the monoclonal antibody 21G2 wasapplied to each piece at a site 10 mm from the bottom, and a solution ofgoat anti-rabbit IgG polyclonal antibody (product of Cappel) at a site15 mm from the bottom, using Biojet Q 3000 (product of Biodot). After 2hours of drying at room temperature, each sheet was blocked by 10minutes of immersion in 1% skim milk (product of Difco)−0.1% Tween20-PBS, followed by thorough drying.

(2) Preparation of a Colored Latex Particle-Labeled Product

a. Red Latex Particle-Labeled Anti-Helicobacter pylori Antibody

PBS (1.2 mL) was added to 300 μL of a red latex particle dispersion(PL-Latex, 10%, 450 nm, product of Polymer Laboratories), and themixture was centrifuged at 13,000 rpm for 5 minutes. To the sediment wasadded 1 mL of a solution of the monoclonal antibody 21G2 (5 mg/mL) and,after thorough mixing, the reaction was allowed to proceed at roomtemperature for 1 hour. For removing the unreacted portion of themonoclonal antibody, centrifugation was carried out at 13,000 rpm for 5minutes, the sediment was suspended in 1.5 mL of PBS, and the suspensionwas again centrifuged. Blocking was effected by adding 1 mL of 1% skimmilk and allowing the reaction to proceed at room temperature for 1hour. Thereafter, centrifugation was carried out ae 13,000 rpm for 5minutes, and the sediment was suspended in 1.5 mL of PBS containing 1%skim milk-0.01% sodium azide.

b. Blue Latex Particle-Labeled Rabbit IgG

Blue latex particle-labeled rabbit IgG was prepared using a blue latexparticle dispersion (PL-Latex, 10%, 450 nm, product of PolymerLaboratories) and rabbit IgG (0.5 mg/mL, product of Cappel) andfollowing the same procedure as mentioned above.

c. Colored Latex Particle-Labeled Product

Equal amounts of the above two kinds of colored latex particle-labeledantibody were mixed up, and 5 mm×5 mm pieces of Bemliese (registeredtrademark) nonwoven fabric (product of Asahi Kasei Co.) were impregnatedwith 10 μl of the mixture and then air-dried.

(3) Preparation of Immunochromatographic Test Pieces

The colored latex particle-labeled product was overlaid on theantibody-immobilizing support to a site of 2.5 mm from the bottom.Further, a carrier (3 MM Chr, product of Whatman) for immersing in thetest solution was further overlaid on the colored latex-labeled productto a site of 2.5 mm from the bottom. A water-absorbing carrier (3 MMChr, product of Whatman) was overlaid on the antibody-immobilizingsupport to a site of 2 mm from the top and, finally, all the memberswere fixed by covering with a transparent adhesive tape, to give animmunochromatographic test piece.

(4) Detection of Helicobacter pylori in Fecal Specimens

Tests were carried out using feces from the six persons (3 positive and3 negative) described in Example 4. A 0.1-g portion of each fecalspecimen was taken and suspended in 1 ml of 0.1% BSA-0.05% Tween 20-PBS.Impurities were removed by 1 minute of centrifugation at 3,000 rpm, and50 μL of the supernatant was dropped onto the immunochromatographic testpiece prepared in (3) at a site on the carrier for immersing in the testsolution. Ten minutes later, judgment was made as to whether a red linehad appeared at the site of immobilization of the anti-Helicobacterpylori monoclonal antibody. As a result, such red line could not beobserved for each of the negative specimens while a red line could beconfirmed for all the positive specimens. With all specimens, a blueline was confirmed. The test was thus successful.

EXAMPLE 7

[Detection of Helicobacter pylori in Fecal Specimens by the LatexAgglutination Method]

(1) Preparation of a Latex Particle-Labeled Anti-Helicobacter pyloriAntibody

PBS (0.4 mL) was added to 0.1 mL of a white latex particle dispersion(PL-Latex, 10%, 440 nm, product of Polymer Laboratories), and themixture was centrifuged at 13,000 rpm for 5 minutes. To the sedimentwere added 0.5 mL of PBS and 0.5 mL of a solution of the monoclonalantibody 21G2 (1 mg/mL) and, after thorough mixing, the reaction wasallowed to proceed overnight at room temperature. For removing theunreacted portion of the monoclonal antibody, centrifugation was carriedout at 13,000 rpm for 10 minutes, the sediment was suspended in 1 mL ofPBS, and the suspension was again centrifuged. Blocking was effected byadding 1 mL of 1% skim milk and allowing the reaction to proceed at roomtemperature for 1 hour. Thereafter, centrifugation was carried out at13,000 rpm for 5 minutes, and the sediment was suspended in 1 mL of PBScontaining 1% skim milk-0.01% sodium azide.

(2) Detection of Helicobacter pylori in Fecal Specimens

Tests were carried out using feces from the six subjects (3 positive and3 negative) described in Example 4. A 0.1-g portion of each fecalspecimen was taken and suspended in 1 mL of PBS containing 0.1%BSA-0.05% Tween 2.0. Impurities were removed by 1 minute ofcentrifugation at 3,000 rpm, and the supernatant was obtained. A 50-μLportion of this supernatant of fecal suspension and 50 μuL of the latexparticle-labeled anti-Helicobacter pylori antibody prepared in (1) weredropped onto a latex agglutination board and mixed up using a sliderotor (product of Eiken Chemical). Five minutes later, judgment was madeas to the occurrence or nonoccurrence of agglutination by visualobservation. As a result, no agglutination was observed with any of thenegative specimens, while agglutination was confirmed with all thepositive specimens.

EXAMPLE 8

[Molecular Weight Determination of the Antigen in Feces by GelFiltration]

Twenty grams of feces specimens provided by Helicobacter pylori-positivepersons (No. 4 and No. 5 in Table 3) were suspended in 100 mL ofice-cooled PBS. Centrifugation was carried out at 10,000×g for 10minutes, the sediment was discarded, and centrifugation was againcarried out at 90,000×g for 30 minutes to give a supernatant. A 1.5-mLportion of this supernatant was applied to a gel filtration columnpacked with Sephacryl-S300HR (product of Pharmacia) (1.5×140 cm, 0.1 Mphosphate buffer, pH 6.5) and 1.5-mL fractions were collected. Themolecular weight markers used were thyroglobulin, ferritin, catalase,bovine serum albumin and cytochrome c. Each fraction was tested forantigen detection by the sandwich ELISA mentioned in Example 2 (3)(monoclonal antibody 41A5-immobilized plate, biotin-labeled monoclonalantibody 8239, ard monoclonal antibody 21G2-immobilized plate andbiotin-labeled monoclonal antibody 41A5). As a result, Helicobacterpylori-specific antigen in feces was found to have a molecular weight of270 kDa.

EXAMPLE 9

[Antigen Identification]

(1) Antigen Purification

A column (10 mL) was prepared by immobilizing the monoclonal antibody82B9 on CNBr-activated Sepharose 4B (product of Amersham PharmaciaBiotech) according to the method described in Amersham PharmaciaBiotech's Affinity Chromatography Handbook. Helicobacter pylori (ATCC43504) cells were disrupted by sonication and subjected toultracentrifugation, and 5 mL of the supernatant obtained (proteinconcentration 4 mg/mL) was applied to the above column. After 2 hours ofstanding at room temperature, the column was washed with 120 mL of PBS(flow rate about 2 mL/minute) and eluted with 130 mL of 0.2 M glycineHCl buffer (pH 3.0). The washings and eluate were respectively collectedas 10-mL fractions and each fraction was measured for absorbance (280nm) and antigenicity. The antigenicity determination was carried out bythe sandwich ELISA (immobilized 21G2, and 82B9 as label) described inExample 2. As shown in FIG. 1, the antigenicity was confirmed in eluatefractions (fractions Nos. 14 to 17) alone.

(2) Purity and Molecular Weight of a Purified Antigen

An eluate fraction (No. 14; 50 μL) was admixed with an equal amount of asample buffer (2% SDS-5% mercaptoethanol), and the mixture was boiled at100° C. for 5 minutes. A 20-μL portion of this solution was subjected toSDS-polyacrylamide gel electrophoresis (4-20% acrylamide gel). Themolecular weight markers used were phosphorylase b, bovine serumalbumin, egg white albumin, carbonate dehydratase, soybean trypsininhibitor and α-iactoalbumin. After electrophoresis, the gel was stainedwatt Silver-Stain KANTO III (product of Kanto Chemical). As a result,the purified antigen gave a single bard and its molecular weight was 59kDa. Further, 1 mL of the eluate fraction (No. 14) was subjected to gelfiltration on Sephacryl-S300HR in the same manner as described inExample 6. The fraction obtained was tested for antigenicity by sandwichELISA and, as a result, this purified antigen was found to have amolecular weight of 270 kDa and to be the same one as the antigen infeces.

(3) Amino-Terminal Amino Acid Sequence Determination of the PurifiedAntigen

Using 300 μL of the eluate fraction (No. 14), the amino-terminal aminoacid sequence of the purified antigen was determined using the HP G1005AProtein Sequencing System (product of Hewlett-Packard). The sequence ofthe eight residues from the amino terminus was found to beMet-Val-Asn-Lys-Asp-Val-Lys-Gln. This sequence is in complete agreementwith the amino-terminal sequence of Helicobacter pylori catalase (J.Bacteriol. (1996), 178, 6060-6967).

(4) Catalase Activity and Ultraviolet-Visible Absorption SpectrumMeasurements of the Purified Antigen

Since the amino-terminal amino acid sequence of the purified antigen wasin agreement with that of catalase, each fraction was assayed forcatalase activity. The reaction solution used was 11 mM hydrogenperoxide-containing PBS. Each fraction was diluted 100-fold with PBS,and the reaction was started by adding 50 μL of the dilution to 2 mL ofthe reaction solution. The reaction was carried out at room temperature.Absorbance measurements were carried out at 240 nm at timed intervals,and the decrements in absorbance per minute were determined. 235 wasused as the blank. As a result, catalase activity was detected in theantigen fractions, as shown in FIG. 1.

For identifying he hem contained in the catalase, the ultravioletvisible absorption spectrum of the eluate fraction No. 14 was measured.As a result, absorption maxima were observed at 407 and 277 nm. Thesevalues were very similar to the literature values for Helicobacterpylori catalase (405 and 280 nm, J. Gen. Microbiol. (1991), 137, 57-61).

(5) Storage Stability of the Purified Antigen

The eluate fraction (No. 14) was diluted to 1.4 μL/mL with 0.1%BSA-containing PBS, and the solution was stored at 25° C. for 1 week.After storage, the purified antigen was assayed for antigenicity by thesandwich ELISA described in Example 5 and for catalase activity by themethod described above in (4). When compared with the purified antigenfrozen stored at −80° C., there was little change in antigenicity afterstorage at 25° C. However, the catalase activity showed a markeddecrease and the residual activity was not more than 5%.

EXAMPLE 10

[Reactivity of the Anti-Helicobacter pylori Monoclonal Antibodies withthe Subunit of the Antigen in Feces]

For estimating the reactivity of the anti-Helicobacter pylori monoclonalantibodies with the subunit of the antigen in feces, 50 μL of the fecalsuspension supernatant from each of the Helicobacter pylori-positivesubjects described in Example 8 was subjected to SDS-polyacrylamide gelelectrophoresis as described in Example 9 (2). As a control, 50 μL ofthe purified antigen of Example 9 (eluate fraction No. 14) was alsoelectrophoresed in the same manner. After electrophoresis, proteins weretransferred to a nitrocellulose membrane by the western blottingtechnique. The nitrocellulose membrane was immersed in 1% skin milk for1 hour for blocking, and then reacted with the monoclonal antibody 21G2or 82B9 for 1 hour. The nitrocellulose membrane was washed with fiveportions of 0.05% Tween 20-PBS, and then reacted with theperoxidase-labeled anti-mouse IgG antibody. The nitrocellulose membranewas washed with five portions of 0.05% Tween 20-PBS and, after additionof a substrate solution (peroxide+3,3′-diaminobenzidine) incubated for10 minutes. Either of the lanes resulting from migration of the fecalantigen and purified antigen did not show any antigenicity-due colordevelopment with any monoclonal antibody. For ascertaining the cause offailure in detecting the antigenicity of the subunit molecule, whether atreatment for dissociation into subunits causes changes in antigenicityor not was examined by the dot blotting technique. Thus, 5-μL portionsof the specimens before and after dissociation treatment with 1% SDSwere dropped onto a nitrocellulose membrane. After air drying, theproteins adsorbed on the nitrocellulose membrane were assayed forantigenicity by the same method as mentioned above. As a result, it wasfound that both the fecal antigen and purified antigen show markeddecreased in antigenicity after dissociation treatment with 1% SDS. Itis therefore suggested that the monoclonal antibodies of the presentinvention do not recognize the subunit of catalase, namely the epitopeof the primary structure, but recognize a more native, higher structureas the epitope.

EXAMPLE 11

[Purification of the Antigen in Feces]

(1) Molecular Weight of the Antigen in Feces

Feces (165 g) provided by a positive subject with the highestHelicobacter pylori antigen level in feces (Specimen No. 8 in Table 4)were suspended in PBS (4 times the weight of feces), and the suspensionwas centrifuged (7,000 rpm, 30 minutes) to give a supernatant, which wasfurther ultracentrifuged (30,000 rpm, 30 minutes) to give a supernatant.Ammonium sulfate (165 g) was added to 680 mL of the supernatant toattain 40% saturation, the mixture was stirred, and the resultingprecipitate was removed by centrifugation (7,000 rpm, 30 minutes). To750 mL of the supernatant was added ammonium sulfate (214 g) to attain80% saturation, the mixture was stirred, and the resulting precipitatewas recovered by centrifugation (7,000 rpm, 30 minutes). The precipitatewas suspended in PBS and dialyzed against 10 L of distilled water. A2-mL portion of the dialyzate (82 mL) was subjected to gel filtration ona Sephacryl-S300HR column in the same manner as in Example 6. Thefractions obtained were examined for antigenicity by sandwich ELISA inthe same manner as in Example 5. The molecular weight of the fecalantigen was found to be 270 kDa, thus identical to the molecular weightof the fecal antigen of the two positive persons described in Example 8and of the purified antigen described in Example 9.

(2) Purification of the Antigen in Feces

A 40-mL portion of the dialyzate described in (1) was diluted to 100 mLwith 10 mM phosphate buffer (pH 7.0), and the dilution was applied to acolumn (1×2.5 cm) packed with a cation exchange resin, CM-Sephadex C-50(product of Amersham Pharmacia Biotech), and equilibrated with the samebuffer. After washing with 10 mL of the same buffer, elution was carriedout with 10 mL of PBS. The effluent, washing and eluate fractions wereeach 320-fold diluted with PBS containing 0.1% skim milk and thenassayed for antigenicity by the sandwich ELISA described in Example 5.Antigenicity was found in the eluate fraction.

A column (2×3 cm) was prepared by immobilizing the monoclonal antibody21G2 on CNBr-activated Sepharose 43 (product of Amersham PharmaciaBiotech) according to the method described in Amersham PharmaciaBiotech's Affinity Chromatography Handbook. A 6-mL portion or the aboveeluate fraction was applied to the monoclonal antibody-immobilizedcolumn. After 2 hours of standing at room temperature, the column waswashed with 50 mL of PBS and then eluted with 0.2 M glycine HCl buffer(pH 3.0), and 10 mL fractions were collected. Each fraction was 10-folddiluted with PBS containing 0.1% skim milk, and the dilution was assayedfor antigenicity by the sandwich ELISA described in Example 5. Catalaseactivity was assayed by the method described in Example 9. As a result,as shown in FIG. 2, antigenicity was observed in eluate fractions(fractions Nos. 7 and 8) collected immediately after the start ofelution indicated by an arrow. Catalase activity was found in agreementwith the antigenicity. In view of the foregoing, this fecal sample wasfound to contain a native antigen having four subunits and havingcatalase activity.

INDUSTRIAL APPLICABILITY

The invention, which has the constitution mentioned above, can providemonoclonal antibodies capable of recognizing the epitope occurringspecifically in Helicobacter pylori catalase. Further, by using themonoclonal antibodies of the invention, it is possible to veryspecifically recognize Helicobacter pylori. Hybridoma lines producingmonoclonal antibodies recognizing Helicobacter pylori catalase havesuccessfully been established, so that the same monoclonal antibodiescan be produced semi-permanently. The diagnosis kit in which themonoclonal antibody of the invention is used can use digestive tractexcreta as specimens and can detect Helicobacter pylori infection in asimple and efficient manner without causing pain on subjects. Even whenonly one monoclonal antibody species is used, the diagnosis kit of theinvention shows very good precision, shows no difference among lots, isstable and can detect Helicobacter pylori infection always specificallyand with great accuracy.

1. A test method for detecting a native Helicobacter pylori catalase infeces which comprises: (a) contacting a fecal specimen from a patientsuspected of Helicobacter pylori infection with at least one monoclonalantibody against the native Helicobacter pylori catalase to form acomplex of the antibody and the antigen; and (b) detecting saidantibody-antigen complex, thereby determining the presence of nativeHelicobacter pylori catalase in said fecal specimen.
 2. The test methodaccording to claim 1, wherein the at least one monoclonal antibody isproduced by at least one hybridoma selected from the group consisting ofhybridoma 21G2 (Deposit No. FERM BP-7336), 41A5 (Deposit No. FERMBP-7337), and 82B9 (Deposit No. FERM BP-7338).
 3. The test methodaccording to claim 1 or 2, wherein said test method comprises an ELISAtechnique.
 4. The test method according to claim 1 or 2, wherein saidtest method comprises an immunochromatography technique.
 5. A diagnosiskit for detecting a native Helicobacter pylori catalase in a specimen,which comprises at least one monoclonal antibody produced by at leastone hybridoma selected from the group consisting of hybridoma 21G2(Deposit No. FERM BP-7336), 41A5 (Deposit No. FERM BP-7337), and 82B9(Deposit No. FERM BP-7338).
 6. The diagnosis kit according to claim 5,which further comprises a means for carrying out an ELISA technique. 7.The diagnosis kit according to claim 5, which further comprises a meansfor carrying out an immunochromatography technique.
 8. A hybridoma whichis 21G2 (Deposit No. FERM BP-7336), 41A5 (Deposit No. FERM BP-7337), or82B9 (Deposit No. FERM BP-7338).
 9. A monoclonal antibody which isproduced by the hybridoma according to claim 8.